ITAM signaling controls the half-life and surface retention of peptide-MHCII complexes. DCs were pulse-labeled with [³⁵S] for 30 minutes, washed, and chased for the indicated time points. Cells were then lysed, and mature MHCII complexes were immunoprecipitated with Y3P antibody before resolution by PAGE and autoradiography. In WT DCs, stable peptide-MHCII complexes (αβ-peptide), which are SDS-resistant in nonboiled samples, appeared by 3 hours and were slightly reduced after a 9-hour chase. While Vav^NULL (A) or DF (B) DCs generated stable peptide-MHCII complexes at 3 hours, they rapidly decayed by the 9-hour time point. (C) Quantification of pMHCII decay from 3-9 hours was achieved by densitometric analysis of pMHCII bands at 9 hours normalized to 3 hours. Data represent the relative mean reduction in pixel intensity ± SD from 4 independent experiments. (D) WT and Vav^NULL DCs were treated with Brefeldin A to block transport of newly synthesized MHCII through the Golgi network, and surface MHCII was detected by FACS at the indicated time points to determine the rate at which MHCII was removed from the cell surface. Data represent the mean percentage ± SD of remaining surface MHCII (based on mean fluorescence intensity [MFI] at time 0) from 5 independent experiments; *P < .05, **P < .01.